Homologous recombination reshuffles genetic information between parental chromosomes generating genetic diversity and driving evolution. Recombination predominantly occurs at a set of genomic locations called recombination hotspots. In most mammals, including human, hotspot locations are defined by the sequence specific binding of the PRDM9 protein, which tri-methylates lysine 4 of the histone H3 at the sites where recombination is later initiated by the formation of DNA double stranded breaks (DSBs). In Prdm9 knockout mice DSBs are targeted to gene promoters and enhancers, which also carry an H3K4 trimethylation mark. Therefore, PRDM9 directs recombination away from functional genomic elements. This role is important as data indicate mutagenic effects of recombination both at the local nucleotide level and in gross chromosomal rearrangements. Prdm9 knockout mice are sterile with gametocytes eliminated shortly after induction of homologous recombination. Nevertheless, some mammals lack a canonical Prdm9 gene. What defines recombination hotspots in such species is currently unknown. To gain essential insights into possible mechanisms we propose to investigate the recombination landscape in two non-PRDM9 organisms and to determine the biological function of the KRAB domain of PRDM9 that may be expressed in these species. We will employ our ChIP/seq-based approach to build high-resolution genome-wide maps of DSB hotspots in (a) the dog and (b) the short-tailed opossum. We will compare these maps to the maps we recently generated for mice and human to determine the common and different features of DSB hotspots and their distributions in animals that have and that lack PRDM9. We will also generate two mouse models to determine the biological role of the KRAB domain of PRDM9. These will include (a) a mouse line expressing the truncated version of PRDM9 restricted to the N-terminal part of PRDM9 without the DNA binding domain (the form that is found in opossums) and (b) the line expressing full length PRDM9 with a mutant KRAB domain. We will evaluate both models with respect to their distribution of PRDM9-dependent H3K4me3 marks, the ability to initiate homologous recombination, the distribution of DSB hotspots, and the ability to complete recombination. In addition to the prominent role of homologous recombination in establishing the general genomic makeup of the population, recombination per se is essential for proper segregation of homologous chromosomes during gametogenesis. Recombination defects, including reduced recombination efficiency and improper placement of recombination events, are invariably associated with infertility, miscarriage and aneuploidy-related birth defects. Beyond the obvious health implications understanding of PRDM9 function is extremely important from an evolutionary point of view as Prdm9 is the only known speciation gene in vertebrates. Our studies aim to unravel the mechanisms that affect recombination efficiency and distribution in mammals, the factors that may interfere with recombination progression, and the mechanisms involved in mammalian speciation.